CA2272793A1 - Porcine transmissible gastroenteritis virus oral vaccine production in plants - Google Patents

Abstract

The present invention provides a method of preparing at least one portion transmissible gastroenteritis virus (TGEV) protein comprising the steps of obtaining a S gene for TGEV that has been altered for optimal expression in the plant, inserting the gene within a vector suitable for transforming a plant, transforming the plant with the vector and growing the transformed plant. This invention also provides a method of immunizing pigs against porcine TGEV comprising harvesting the transformed plant to obtain harvested tissue and feeding the harvested tissue to pigs, or extracting the protein from the harvested tissue and administering the protein to the pig as a food supplement or as an injection. Direct grazing of the plant tissue prior to harvesting may also be employed as desired. The invention extends to isolated DNA
molecules encoding a S gene for TGEV that have been altered for optimal expression in the plant and to vectors comprising these genes.

Description

_ . .. _.... ", , , ,:~. , . ~ . "~..,~..,~.~.~, ~. r;~.t Title: PORCINE TRANSMISSIBLE GASTROENTERITi~S VIRUS ORAL Vtlf'f_'t PRODUCTION IN PLANTS
The present invention relates to the production of an animal vaccine in plants and to t;~r~rs and vectors for the expression of the vaccine. More specifically, this invention relates t:e expression of at least one gene obtained or derived from a porcine transmissible gasiracrani~ a virus in plants. This invention also relates to administration of the transgenic plant, ar E~rc':.: .:~
isolated therefrom, to induce the production oFprotective antibodies in pigs.
to BACKGROUND OI< 'THE INVENTION
Transmissible gastroenteritis (TGE) of swine is a coronaviral infection causing severe; of~~r~
fatal diarrhea in young pigs. Due to the rapid development of the disease, the u~osi susceptible newborn piglets are usually unable. to develop their own protective immunity against the vircis.
i s Their survival depends on the presence of TGEV specific antibodies in the colostrum and milk :af the sow. The type of these antibodies is very important. The most effective protection is provitfect by secretory (s)IgA molecules capable of surviving for extended periods of tune in the dig:aa~c tract. Mucosal immunity to TGEV is crucial in preventing infection, not only because it I;rot~,:;ts the sow but also because the (s)IgA producing plasma cells are transferred tbro::l:h :iii 2o immunological bridge into the mammary glands providing protection for the suckling l.~i'~;leaC as well.
Protective immunity is directed to the spike (S) protein of TGEV [Ganves et al., 1 ~%?8, limenez et al., 1986]. Neutralizing antibodies recognize well-characterized epitopes on the am~;~o-25 terminal domain of the S protein. Four major antigenic sites (A, B, C and D) are known in this ~~art of the protein [Cornea et al., 1990, Jimenez et al., 1986]. Epitope A and, to a lesser extern P, :.;re involved in neutralization and it has been shown that in order to achieve a protective icrun:ine response equivalent to that induced by the full S protein, the intact globular N-tt:rminal hair is essential [Tuboty et al., 199].

Several attempts have been made to develop recombinant TGEV vaccines. 'I'hese vac~~nes must not only fu1611 the general vaccine requirements, but must also be able to induce a ~~u~c>sal immune response, in particular local virus neutralizing sIgA antibodies. The S
gene has h:en expressed in vaccinia virus [Hu et al., 1985], baculovirus [Godet et al. 1991, Tuboly et a: ;a'r':]
35 and in a human adenuvirus [Tortes et al., 1996]. It also has been expressed in some prc~k:!r5'~~tic vectors [Smerdou et ai., 1996].

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2 Dramatic evidence of the utility of plants as bioreactors was presented in two recent pap~rrt, the first of which demonstrated that the four chains of a secretory immunoglabulin wer:: l~rcsper_-expressed and assembled in plants, and that the antibody was fully functional [1~1a et ~:L, ; 9ie 1.
Other work has extended the concept to humari haemoglobin, and further confirmed the ,ititiy t~-f plants as bioreactors. These examples have been based on Agrobacterium mdeaf:-:.i transformation, which allows the development of stable transgenics _that can be reproduced fr; ;,~
seed as required.
to Recently the fill S gene and the amino terminal domain coding sequenc:4 maw e~~.
expressed in an arabidop~is plant system [Gomez et al., 1998]. Although this study shov.~c~1 tl;;.
feasibility of expressing the mainly glycosylation dependent S proteins in planes, tl~o ext,r L.;:.i:-~n levels were not signific,int as the transgenic protein could not be deaectcd.
The only ir~~-Iir~:.t evidence of expression was given by a positive immune reaction in transgcnic plait prot~~i~n injected mice. Another report describes methods for the expression of the full length TGt~.'V .~,v _e protein (E2) in corn [Howard, 1997]. No data on protein expression levels or immunization a:cd antibody production in pigs is provided. There remains a need for an efficient mothc~d f~3; t-i~~
expression of TGEV protein, or fragments thereof, in plants.
2o SUMMARY OF TIIF d>~ VENTION
The present invention relates to the production of an animal vaccine in placits arid to ve;~::
and vectors for the. expression of the vaccine. 1\~Iore specifically, this mventioi~ Te~a~as ':ic' expression of at least one gene obtained or derived from a porcine transmissible gaslrcicnteriis virus in plants and the administration of this gene product to pigs.
According to the present invention, there is provided a method of preparing at lea:,t f?n porcine transmissible gastroenteritis virus (TGEV} protein comprising the steps of obtaini;tg a S
gene for TGEV that has been altered for optimal expression in the plant, inserting the gene ~~ith«: a 3o vector suitable for transforming a plant, transforming the plant with the vector and growin, ~~e transformed plant. Furthermore, this invention is directed to the above zmethod wh;,rc:ici ~.;e.=, '~; ~;=v,~
for TGEV is truncated at the 3' end by approximately C~00 base pairs or vvl~crein tEe 5 g~.t~.e .as been resynthesized for optimal plant codon recognition, for example, the synthetic ~~r~~~ ,hmA~), i;~
Figure 1.

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3 The invention also provides a method as described above, wherein the plant is any hlart tl;:
is useful as animal feed, for example alfalfa, barley, corn, flax, soybean, sunflower, rapES~=>i, wheat and the like. Preferably the plant is alfalfa or soybean. Also included within the scc.~pe a:f the present invention is the method as described above, wherein the plant is any plant useful 3~~
study the expression of proteins in plants, such as tobacco.
Also considered within the present invention is a DNA molecule as shown in Fif~ure 1 is~.~.r a vector comprising this I7NA molecule operatively associated with promoter, ~.~i~a;=e~~ _:y terminator regions. Furthermore, the invention provides plant cells, plant seeds and W~c~i~ p 1;:~" ~;
to comprising the above defined vector. The invention also provides a vector comprisit~~ a 'f(ic'-J v, gene that has been truncated at the 3' end by 600 base pair operatively associated wlLh pJ'oFT:~?i~_', enhancer and terminator regions as well as plant cells, plant seeds and whole plants cornprisirty t~~~s vector.
This invention is also directed to a method of immunizing pigs against transmissilri~;
gastroenteritis virus comprising transforming a plant with one of the vectors as defined ab~-TI_, growing the transformed plant, harvesting the transformed plant to obtain harvested tissue, fez.dir~g the harvested tissue to pigs and repeating the steps of feeding as neederi.
Alst~ included within the provisions of this invention is the method of immunizing pigs as defined above wherein t;i~
2o harvested tissue is extracted and the extract is used to immunize a pig.
This immunization n~a:e:>r achieved by any suitable means, for example injection or oral delivery of a composition cornpri=ag an extract and a suitahle adjuvant. The invention also extends to the administration of t.c~a~~
harvested transgenic plmt tissues, obtained according to a method of the invention, to a pig.
The invention also provides a method of immunizing pigs against transmi,sitylc gastroenteritis virus comprising transforming a plant with one of the vectors as defined abo~~~z growing the transformed plant, harvesting the transformed plant to obtain h~t~ested tit.t:_~, extracting the a protein encoded by the DNA molecule present within the vector from the har:~es::d tissue and administering the protein to the pig as a food supplement or as an injection.
;
In a further aspect of the present invention, there is provided an immunogenic comp,-.vs~ti::zt comprising a vaccine antigen that provides protection against porcine transmissible gastTOe;ate;=ti:=
virus. According to this aspect, a transgcnic plant is transfonned with one of the ve~:.tors as described above. The vaccine antigen is then isolated from the plant and incar~orat~d ir~tt-~ a vaccine composition.

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4 BRIEF DESCR~PT10N OF THE DRAWINGS
These and other features of the invention will become more apparent from the fcllc~;y~~~
description in which reference is made to the appended drawings in which:
Figure 1 shows the nucleotide and amino acid sequences of a synthetic TGEV S
gwne that lraw 1~~: rt modified for optimal plant colon recognition. The coding region for the tobacco Pig siLnal h~~~ios at nucleotide number 9 and ends and nucleotide number 74, followed immediately by tl~e ~.c;~._v;:.g region for the S protein. Underlined regions are the restriction sites for Xhai :uml a~i. 'il~.
t o underlined fragment at the N-terminnrs of the amino acid sequence is tl5e signal pclati;3c ;~ f tf~l~"~; ;~
pathogenesis-related protein. 'fhe italized sequence at the C-terminus of the amino acid s~~;u~:vc.~ a a His tag.
Figure 2 is a diagram of Gene constructs transferred to plants in binary vectors according to t==~
t5 methods of the invention. NPT-II is the plant selectable marker regulated by the ?~it7S pror,~ot,r (NP) and terminator (NT). Expression of the S gene was regulated by the Super promoter and N~.'.-S
terminator. The S gene portion of each construct is indicated above by the arrows, the numhcas representing the nucleotide positions within the coding region of the S gene, with 1 as the first lraae of the ATG colon. In pSS 1 the SP is the tobacco signal peptide coding sequence and t:ee I
zo represents the 6xHis-ta.
Figure 3 is a schematic representation of the procedure used in the synthesis of a TGF.V ~~:vi that has been modified f'or optimal plant colon recognition 2.5 Figure 4 is a Western blot confirming transgenic protein size (expressed in tobacco), wherein L.acie l: TGEV, Lane 2: L)ninfected ST cells, Lane 3: pS4, Lane 4: Non-transgenic plant, Lane 5; p5~:1, Lane 6: molecular weight marker (in kDa).
DETAILED DESCRIPTION OF THE INVENTION
30 , The present invention relates to the production of an animal vaccine in plants and to t;~~--~;
and vectors for the expression of the vaccine. l~lore specifically, this inventu~n relare.-s t .e expression of at least one gene obtained or derived from a porcine transmissible gastrocr~ieritis virus in plants.
~5 . , ,... ... .... ,~~ .,~.", ~,m ii,t.. v.t'. tin.:,t..n<s,H ~~:sin The development of plant based oral vaccines (edible vaccines) offers a new approa::l2 to vaccination strategies, especially in cases where local intestinal immune response is crucial t:3r tl=s~
prevention of infection. Major advantages of plant-derived edible vaccines include the Inw cost :~f production and the ease of delivery to an animal. The immunogenic proteins are produced in t:~

5 plant and the plant material containing the trarisgenic protein may be delivered to the a=I~ma!s :;
part of the regular feed. Porcine transmissible gastroenteritis is a typical intestinal disease vv;i severe consequences generally leading to the death of young piglets.
Rerlication of thY~ vir?i~ :s restricted mostly to the small intestine where it destroys the intestinal cells resultir~; ir: a l~~;a. ~.
watery diarrhea. Although the virus is capable of replication to a lesser extent in oth:,r or' ._:_; ";:j replication usually occurs without consequence. Progression of the diarrhea is so rapid th,et t;i~r~ ;
no time for other symptoms to develop. Development of the intestinal disease can a~i ~ i a prevented by the presence of a sufficient amount of virus neutralizing antibodies in the sr;~~il intestine.
According to one aspect of the present invention, it is shown that the S gene of TGEV cz~n be expressed in different forms in tobacco and alfalfa plants at a higher level than faund m r~_y previous reports. Tobacco (Nicotiana tabacum) is a convenient "test" or "model" crop, especially for testing expression in vegetative tissue. All constructs were driven by a strong synthesis promoter, the so called "super promoter" [Ni et al. 1995], which is considered to coul~~r 2o constitutive expression.
In an attempt to improve the TGEV S gene expression in plants, vector pS4 containing =re cDNA of the original S gene, but truncated at the 3' end by approximately 6U0 bp, was expresscci in tobacco plants. This cDNA clone has previously been shown to be equivalent to the intact clone irx the induction of neutralizing antibodies [Tuboly et al,, 1995]. While not wishing to be limit:~d '~y theory, it is believed that deletion of the 3' end amino acid residues remove the part of the S prt~teir~
which anchors it to the plant membrane, permitting it, therefore, to diffuse into the apaplast, wh~a~e it is expected to accumulate to higtrer levels and interfere less with the plasrnalcmlna. Tl~a constuct, labeled pS4, is illustrated in Figure 2.
;
Another strategy for enhancing the expression of the TGEV S gene in plants invol::.d changing the nucleotide sequence of the S gene, leaving the amino acid sequence ir~ta~t. A detai;;d computer analysis of tl-ie cDI'TA sequence of the S gene indicated several features that ma~.~ ~-~e problematic for expression in plants [Koziel et al. 1996]. There are at least

6 potential pla~rt ~~i:~i:w-~
signals distributed throughout the sequence, as well as several mRNA
destabilizing sf:;~aencw.. _;v:.h as ATTTA. In addition, the AT content is over 65°/~, there are codons which are urstahlL. irz lj?:~~.-.ts, . _ . . . . .. ,_ , .,.... . ,~n ~ m. n, v . r . nt~..m.:w<a.rr ;L,;,pc:
..

splicing sequences, and many codons atypical of plants. From extensive analyses of s~v;~~..1 hundred plants in earlier experiments with the S35 promoter (unpublished) it was cc~ncluclerl t'r at changing the promoter alone would probably not be enough to overcome the low exprcssian =_~fi r.;:
native S gene in plants. To address these problems, it was decided to synthesize the first 1- ~~s ?;o s of the S gene, rather than attempt to modify the existing sequence by site-directed r~~utal,tvr~c; :s.
This section of the S gene contains all the neutralizing epitopes of the protein. In additin-r, lre native secretion signal was replaced with a tobacco PR signal peptide [Van Kan et zl. lllEr~], a=i,..
6xhistidine tail was added to facilitate purification of the protein from plant extracts, The. pr-;a'r-r produced from this construct was expected to be analogous to the naturally c~~cl:,.rriry firm ~-r, to except for glycosylation components. This construct, labeled pSSI, is illustrated .in Ni~;r~re ~ r=_~vd the nucleotide sequence of the of the synthetic S gene is provided in Figure 1 al=~n5 w-irr, corresponding a.rnino acid sequence. A schematic representation of the pro~c~turre es=.ci ttt synthesize the modified S gene is provided in Figure 3.
is As a result of the above modifications two lines of transgenic plants were generated aT;d t~.r.e gene expression levels of both the pS4 and pSS 1 constructs proved to be higher than the expression levels reported earlier. The recombinant proteins were detected in Western blots whirr av.,o confirmed that the size of the proteins was as expected. Based on comparisons with lr;~=~~~n amounts of S proteins, the proportion of the transgenic protein was calculated and fi~und ;a y;H~.r 2o between 0.1 and 0.2 °~o of the total soluble protein of the plant tissue. This is 5 to 10 ti=, es lv=~.a-._;
than the results reported earlier for pl;utt expressed TGEV S l;enes [Gomez et al., 1998].
The immunogenicity of the transgenic proteins was tested in pigs, the n<~tural host oC
transmissible gastroenteritis virus. These immunizations also confirmed that a higher level o!~
25 expression was obtained by modifying the gene as described above. Despite the low doses r~~oc3 the use of a less aggressive oil emulsion adjuvant only once, measurable TGEV
specific arrtibauie:
were obtained in the piglets. Although the number of piglets injected (3 in each group) dc~os riot allow for statistical analysis, it is clear that there is a difference among the immunogenic capa:_=t_y of the constructs. Injections with pS4 proteins resulted in slightly higher VN
and ELISA tit~~.rs t'=.=.arr 30 the injections with pSSl proteins. While not yvishing to be limited by theory, this is proba'~ly :a,_~
to the size of the protein and to the fact that the immunologically most important half of tire .' is completely present. The redesigned gene in pSS 1 also induced both VN and ELISA dete:~.'a.!~le antibodies with expression levels that were similar to that of pS4.
35 Assembly of the synthetic TGEV S gene of this invention is performed using stw~d:~rd technology used in the art. The TGEV S gene, designed for increased expression in plant<: is . . .m Iv.Y-f 1.1.v n ,m. ,)c:l .)G,.)V (1~~~~
assembled enzymatically, within a DNA vector, from chemically synthesized oliganucleotica duplex segments. The symthetic TGEV S gene is then transformed into plant genomes using methods known in the art. It is contemplated that a transgenic plant comprising the heterol~~g~~as protein may be administered to an animal in a variety of ways depending on the need ar,d I r;
situation. For example, if the protein is to be orally administered, the plant tissuo ntaa he har~-e:;t-d and directly fed to the animal, the harvested tissue may be dried prior to feeding, or ihc~ animat n~s:y be permitted to graze on the plant with no prior harvest taking place in countries wlre.-.rc t'.n~ i~. s~;, of the feeding practice. 1t is also considered within the scope of this invention i;~r tl~~,: har:-e,,5,-,1 plant tissues to be provided as a food supplement within animal feed. The harveste:l tt5sl~e ~;;':~~~
to also be extracted using methods known in the art and the extracts used, either diret.tl;~ er as i~.rt r:f a composition, for immunization of the pig. Furthermore, the protein obtained from the trars~~_i~
plant may be extracted prior to use as a food supplement, in either a crude, partially puri~?n :~r purified form. The administration of any of these protein fornls to the pigs results in the format;;,I~.
of an antibody that protects the animal from TGEV.
l5 Plant cells and tissues to be transformed include those plants useful as animal fend suc'~ z5 alfalfa, barley, corn, flax, soybean, sunflower, rapeseed, wheat and the like.
The preferred plni species are soybean and alfalfa. Also useful are plants which may be used to study expression of proteins in plants, such as tobacco.
The term "promoter" as used herein refers to the nucleotide sequences at the 5' cncl ~e~ a coding region, or fragment thereof that contain all the signals essential for the initiation c;f transcription and for the regulation of the rate of transcription. The promoters used to ~;xei~pl;'a this invention are constitutive promoters that are know m to those skilled in the art. By "constit;::~r~
promoter" it is meant a promoter that directs the expression of a gene throughout the various parts of a plant and continuously throughout the plant development. Examples of such promoters llns:-.vr) in the art include the synthetic super promoter [Ni et al. 1995] and the CaMV
35S pron-fot~r. The preferred constitutive promoter is the synthetic super promoter. If tissue specific expressio<~ c~f~ a gene is desired, for example seed or leaf specific expression, then promoters specific to tla~se 3o tissues may also be employed. ;
Furthermore, as would be known to those skilled in the art, inducible pron.ctcrs rnay nl~~-. ha used to regulate the expression of the gene following induction of expression b; llro~>ic4isy~ _lt:.
appropriate stimulus (inducer) for inducing expression. 'The use of inducible pron~ot~:ns r~as:~ L;:
required as constitutive synthesis of a foreign protein may, in some cases, be toxic or inrihit =~oin:.rl growth of the plant, v~: ith the result that the only plants which regenerate following exr~ressior are ", , ~ . YY 1 .v , ~ ., , .~ ,,~ i .,~.tU nlw I \,I~. \ . f'.
IlI~,51~;:11t(:II ~~' l; I l ,, 8 , those expressing low levels of the foreign protein. In the absence of an indu:er, the D ~- ~~
sequences or genes will not be transcribed. Typically, the protein factor that bind: spe;;if callv tc an inducible promoter to activate transcription is not present or present in an inactive fcrrn ~rict i.=.=.
then directly or indirectly converted to the active from by the inducer or inducing tr~atmLr,.i. 1' inducer may be a chemical agent such as a protein, metabolite, growth regulator, herbici:-le ot~
phenolic compound, a physiological stress imposed directly by heat, cold, salt, 17~r-pest ot- t~r:c elements or indirectly through the action of a pathogen or disease agent such 1s a virus. A ~ 1: _zt cell containing an inducible promoter may be exposed to an inducer by extemal!y ;:x ;,i;~r; '<_~
inducer to the cell or plant such as by spraying, watering, heating or similar methods.
to To aid in identification of transformed plant cells, the constructs of this in~.~enticm rn,;;y- ~?c further manipulated to include plant selectable markers. Useful selectable markers i~~ti~l enzymes which provide for resistance to an antibiotic such as gentamycin, hygromycin, k~.ttalr,y;..i:~
and the like. Similarly, enzymes providing for production of a compound identifiable by ccflL~ur t 5 change, such as GUS (~i-glucuronidase), or luminescence, such as luciferase, are useful.
By "transformation" it is meant the stable interspecific transfer of genetic information ;taat is manifested phenotypically. The constructs of the present invention can be introduced into t-,;:utt . cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation, rnio:-~~-2o injection, electropnration, etc. as would be known to those skilled in the art.
The term "isolated" as used herein refers to a nucleic acid molecule substantially fret; of cellular material or culture medium when produced by recombinant DNA
techniques or cherr~i:w:
precursors when chemically synthesized.
Also considered as part of this invention are the trangenic plant cells, plant seeds and ~x~t~~le plants containing a c.himeric gene construct of the present invention. Methods of regcnaratug whole plants from plant cells are known in the art, and the method of obtaining transfnmcd :~r~d regenerated plants is not critical to this invention. In general, transformed plant cells are ct~lt~~~,~1 in an appropriate medium, which may contain selective agents such as antibiotics, when= seiect::b'c markers are used to facilitate identiFcation of transformed plant cells. Unce callus forms, ~luc~l formation may be encouraged by employing the appropriate plant hormones in accordance ~-=itl~
known methods. 1n some circumstances, shoot formation may be induced directly fr,~ro transformed cells. The shoots arc then transferred to rooting medium for regeneration of pia:~t,.
The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation. techniques.

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The presence of the DNA sequence coding for the TGEV antigen and expression of tlm TGEV antigen in the transgenic plant can be determined and quantified. An expression ca~sei!::
encoding a TGEV antigen is preferably stably integrated into a plant cell genome. Cta!?li:
s integration of an expression cassette into a plant cell genome can be established »~hn ro~i=~:~ a three successive generations. Methods for the detection of expression of a protein coded tf.~r 'by ~ll inserted DNA include SDS-PAGE, Western blot, EL1SA and other methods known in the a.rt. 'i lxv presence of the DNA sequence coding for the TGEV antigen in the plant genome nt c'~~c.~r~>o~~:~tn;a material can be verified ar,d copy number can be quantified using hybridization ~rl:,t)~ocis kno;=.;3 t:~
to those skilled in the art. The level of gene expression can be quantitated using quantitative ~~~st;:-blots or by measuring the amount of specific mRNA synthesis. Transgeruc planis that are expressing the most TGEV antigen as a percentage of total plant cell protein are preferably selvct~_c1 for further propagation. 'these plants are preferably expressing the TGCV
antigen vi~ithin the ~w=li~,e of 0.1 to 10% of the total plant protein.
Transgenic plants, plant organs and seeds can be combined into animal feed using m:;the:ls and feed components known to those skilled in the art. The amount of transgenic plant, plant u:~::ji~
or seed material added to the feed material is that amount that provides sufficient vaccine antis ~r; ';:
a pig to immunize and:'or protect against TGEV. The amount of "vaccine" will vary depenclin~ .~r 2o the frequency of administration.
Transgenic plants, plant organs or seeds containing a TGEV vaccine antigen can prQ=.:i:~~_ a low-cost, easy-to-administer-and-distribute vaccine composition. The immunogenic v acc.~,~_ composition may be administered orally to pigs. Vvhile not wishing to be limited by thcoy, it is believed that a vaccine a~~tigen administered in a transgenic plant or seeds can immunize aniittals as they chew at the oral mucosa including the tonsils. In addition, it is known that some of the; ani:a:al feed can pass through the stomach to the intestine undigested or partially digested so that mur:-~:,al tissues in the intestine can be exposed to the vaccine antigen. The appropriate range or dc~:~~ csi transgenic plant material and seed can be determined using standard methodology. The range= of 3o doses may be about O.t)1 to 50 mg/kg for oral administration. Once the amount of vaccine. antil-en in the transgcnic plant or seeds is detern~ined, the amount of transgenic plant or seed maieria! t~b~=
administered can be determined.
The transgenic plant or seeds can be administered by feeding to pigs in one or cr~ore L)ssc:~t:
doses at various time inten~als, for example, daily, weekly, monthly or can be feet conti.mr:~-sly.
The development of protective immunity can be monitored by the development of specil=<c tgA

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l0 and/or neutralizing antibodies to the vaccine antigen or a decrease in symptom: i~r rc.o~al~?~:
associated with the infection by TGEV.
The vaccine antigen may also be isolated and purified from transgenic plants and for s:_tuv s using standard chromatob~raphic methods. The vaccine antigen can then be used to in ~r_~atn:r._ animals to provide active or passive immunity or can be used in diagnostic assays.
The following exa~oples, while illu:.arating the embodiments of the inventiotz, ~rv riot to k°=
considered as limiting the scope of this invention in any manner.
to EXPERIMENTAI. E?G11~'IPLFS
Materials and methods t 5 Virus and cell culture The cell culture adapted Purdue 1 l5 strain of TGEV was propagated in a continuous ;~:i_~4 testicle cell (STC) line as described earlier [Tuboly et al., 1994].
zo Construction of trap sjer ~:c~ctors The preparation and cloning of the full length TGEV S gene cDNA has been descril~:~B
[Tuboly et al., 1994]. The DNA was digested with the appropriate enzymes and cloned into t~~e binary pBISNI plant transfer vector behind the synthetic super promoter constructed by Gelvin and 25 co-workers [Ni et al., 19°5]. The final constructs are summarized in Figure 2.
pS4 The S gene cDNA was released from the baculovirus transfer vector TBl-1 6916 [Tuooiv et 3o al., 1994] by a BamHI-XbaI double digestion and cloned into the conrespondinj sites csf !he pBISNI transfer vector. The resulting vector contained a 3.78 kb fragment of the rt-iginal ;~ gt.=ie.
starting 10 basepairs (bp) upstream and ending at 3779 by downstream of the S
gene ATG signal pSS~

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RF:S(:.111('li ~ 1 An 1734 by portion from nucleotide 49 to t».rcleotide 178? of th: or3~'wnal °: :~»n- ~:_--,;,.-...._ was redesigned for optimal plant cedrn -~"~~ivirn. The 'ene was rest'-e~u::;;~;v::.., ._ ... _ . ___ _ .
175-180 by oligonucleotide fragments later Baled tc each other ar,d se:~r,~~:~: _:.~ ; _ _._. .__ _ mismatches. The result~.np synthetic J'~;:1, ;~rwkin~ the S protein sign?
p~,~~~i~~ ~~;_=: = .. . _. ..
S was cloned in frame dowrestream of the a tobacco pathogenesis-related protein cuo'in~~ s;;~;:r:.r__v~ 5 bp) [Van Kan et al. 1989]. An 18 by His-tag coding sequence was adcir_,u tc th;; 3' :.rt:an:; 'l:~= .~:_w was cloned into the Xbal-SstII sites of the pBISNI vector.
Plant material and growth conditions to lc Tobacco and alFalfa plants were grown in ~.:reen house c~~T.:litit.,:;:~
L_u~itl; a da.::'_~ -~.t temperature of 23°C and 19°C respectively) with supplemented lisht to provide a ;a:irY-..-.-..-.~_--a photoperiod of 16 hours. Regenerating shoots were planted in a commercial pottiit~ L~: -:1 fertilized weekly with a 20-10-10 liquid fertilizer. -Production of transgenic tobacco and alfalfa Transfer vectors pS4 and PSS 1 were used for tobacco plant transformations by the Agt-oha~tz-~c~;:_-mediated method. The vectors were transferred to Agrobacterium tumejaciens strain i 58c : r ~h 20 [Koncz and Schell, 1986] containing a helper plasrnid pMP90, by the freeze-thaw method [_~io' ,~:r~
et al., 1987]. Sterile leaves from 4-week old tobacco, cv.Petl-I4 were used for transformaiinn =::a,_:' on the method of Fisher and Guiltinan [1995]. Kana.mycin (300mg~'I) was added to the ~e-at:
medium as a selection agent to eliminate non-transgenic plants. Transgenic plants wee. =u-~':~;
confirmed by PCR using primers based on the inserted genes.
zs Transfer of the transgenes to alfalfa (Medicago sativa), was performed as in Du et al. (1394).
Briefly, petioles of alfalfa were cocultivated with Agrobacterium and regeneration was fro:r. ia:l:~_ via somatic embryogenesis. Regenerating shoots were transplanted to the greenhouse and gro;,r under the same conditions as tobacco.
Southern blot To confirm the presence of the desired genes in the transgenic plants, total piaat DNA u::
extracted by SDS/proteinase K digestion, phenol!chloroform extraction and eti':wcul pr:.:ipi:._ti~;::-:
[Conrad et al. 1995]. The DNA was double digested with the enzymes used fcr insetting the J~.=~-:.

(!fi 11 % 99 f~'R ( l 2: ~6 H~:1\ 1 ;i I S~ 521 :~2;iti (il l I ''I~: \ I'.
R1~:51~: 1R(.'11 the DNA fragments were separated in 0.8°,-o agarose gels, transferred to ~;t:a:z ?:-~~:~~t:;zr.:_=,~-probed according to Sambrook et al. [1989] with the'ZP-labelled TGEV S gene. s ho _:r l~a~?'; '-._ carried the gene of the appropriate size were subjected to further analysis.
Detection of S protein in plants EGISA
The transgenic plants were tested in a TGEV specific ELISE fur the prese,ce of th~:
protein.
Antigen preparation: 100mg leaf tissue from each transgenic and a wild ;yj~e z~r:.:v v-.,:::
powdered in liquid Ir'Z, ?OUuI of TE buffer with 1 % sarcosyl was addod to the pcs~-:~~._-, fra,;;:;- a-a thawed 3 times and incubated at 37°C for 1 hour. The lysate w-as peli;aed iu an yy-~p<-c!:_=.:f centrifuge for 5 minutes at maximum speed. The supernatant was collected and stored at -''!i'r:
until needed.
ELISA: The antigen was diluted twofold in PBS from 1:10 to 1:160, 100 ~cl of.°ac.h di;a=i::f~
was used for the coating of 96 well EL1SA plates (overnight at 4°C or 2 hours at 3 7 vf'). T he ~:=:y was done at room temp=rtore. The plates were washed 3 times with PBS
containing 0.1 °.~° Ty-: ~_:-80 (PBS/Tw). To reduce background, the remaining binding sites were blocked by incubati n~. ~=
plates with a blocking sol:ition (PBS, 0.~'~l NaCI, 0.5°~o BS:~, 0.1%
Tween 20). TtTE~' srec~ic :v:y or rabbit hyperimmune serum was used for the detection of the S protein. Serum and coi=_:~~;__~
dilutions were done in the blocking solution in a separate plate. The serum was d~ttlied te:.=~;~
from 1:10 to 1:10000 and the specific alkaline phosphatase or HRPO labeled conjugatL was diluv~i 1:5000 and used with the appropriate substrate. The plates were washed 3 times af?:.r each with PBS/Tw. The colour reactions were measured in a spectrophotometer at wav~I::n~11 depending on the substrate. Those samples were considered positive for the S
protein that ga~w ~u:
optical density (OD) greater than 0.1 and at lea..st twice the mean OD
obtained with the ne~.a-.>~
sample.
Western blot Western blot analysis of the transgenic plants was done as described by C on r:~d c: ::1.
[1995]. Briefly, 100mg leaf tissue was homogenized in liquid N2 and the proteins separated :a;r~'vr reducing conditions by a 10% SDS-PAGE [Laemmli, 1970]. The proteins were ~ransi2:ie:' e~=
nitrocellulose membranes. TGEV-specific pig and rabbit polyclonal antibodies wcr=.; used tc~ d::=~.c?
the proteins in 1:500 dilution as described, the reaction was detected by the Boehringer ~=~a~lve:<::
chemiluminescent detection kit according to the instructions of the manufacturer.

r)~; I i :'9g HRi 1 2: 47 1-':\.\ 1 .; I ~) 'i:' 1 :~'?;ifi (11 ( I c'I~; \ .
!'. IZI~:SI~: \W'fi ~' ~ ci Swine immunizations Pigs were immunized with transgenic and wild type plan: '..a.re~a~a~:::;
°:, :v v __.. ..
immunogenicity of the recombinant S proteins irf pigs.
Fifteen Yorkshire piglets from a specific pathogen-free herd werw t~':.~u:~~
i8 tv~--. ,_'-.:_ birth and divided into S groups. One group was mock immuni2ed, one group rec;;-~.~:3 w'Iia t'--~
plant protein extracts and three groups were immunized with the transgenic proteins. I'a~l~ '-:_r received one injection of plant protein extract (or PBS in the mock immunized group) v;itl~ an ::il adjuvant at week 0 and two booster doses at weeks 2 and 4 without the adjuvant. T:~e it:j::~:i::::=
were given intraperitoneally. Antigen: 6g of leaf tissue was frozen i:: liquid N~. p:y~:.:r~r~d -:-::
collected in Eppendorf tubes. After thawing, the preparations were centritiyed it an Epp;.°:c?z~vf centrifuge at maximum speed for 5 minutes at 4°C. The supernatant w,~~
~t~ll~:aed-end dil;i'~-vf t-~:-_i:
an equal amount of phosphate buffered saline, pH7.3. Adiuvant: the antigen of the 'rst :ni;:~':_::: n was mixed with an equal volume of an oil emulsion adjuvant (5ml antigen, 4.46>':ul m:r:erai ;~':, O.SmI sorbitan monooleate, 401 polysorbate 80). Blood samples were taken beic.~r4 tl~;. ._.:a injection and weekly throughout the experiment.
Detection of S specif c swine antibodies Sera of the blood collected weekly were separated and tested in a TGEV specif c ELISA :3rd a virus neutralization microtiter assay.
ELIS.4 The TGEV specific EL1SA of the samples was done on TGEV infected and uninfected S" :'.
by a fixed cell assay as described earlier [Tuboly et al., 1993].
Virus neutralization The samples were heat inactivated at 56°C for 1 hour, diluted twofold in DMEh'g and iac:;b:ai::d with 100 TCmso of TGEV P11~ fo: 1 hour at room temperature. The virus and so::;n~ ,~:n:~;t::-::j were added to swine testicle cell monolayers in 96-well plates and incubated for 1 hour .~: ?%v.'.
After the incubation the supernatant was rcmov ~d, tho cells washed with L~~r:lE'~~I a::;re?i li~'~-i F::-i ~'~ I 1: 99 f~Rl 12:47 1:11 t ~I!~ 821 ;>~'36 0l l ft'I~: v.Y. RI'.S1~:.1R(:ll with 5%FCS was added to the monolavers. The evtottathic effects ~.~~ero de;~c:e~? b°~. a i:u~c:::-w and by crystal violet staining 36 hours after the inoculation.
Results Transfer vectors and transgenic,nlants The structure of the transfer vectors is shown in Figure 2. All vectors c.~nt.:ined th-.; ka:,"~a ;> ~.-;
resistance gene driven by the NOS _promoter. The S gene in each case was driven 't>~~ the '~~;o::-to promoter. The pS4 vector contained a 3' tnmcated S gene and pSS 1 ways built un fror=~ 3 rlia~:~r d=a parts: a plant signal peptide, the redesigned S gene portion a:nd a His-t<3 The transgenic plants resistant to the selective medium did not show any morhholc,Vical diff:_rc:~.;, compared to non-transgenic tobacco plants. Approximately 40 different lines of ea.ctyke :~~::_ selected and grown in a greenhouse. Most of these plants were positive for the trattsgene in P=~'R ~-r 15 Southern blotting.
Detection of transgenic S proteins Those plants that were positive in TGEV specific PCR or Southern blotting w°re s~tl-;~ct_:~
z0 to an ELISA assay to detect transgenic protein production. Although the ELISA utiiize~d in T he~
study was not very sensitive still we were able to detect the S proteins produced by the plants. ~ aL
best results were obtained with 1:40 dilution of the plant antigens and 1:1000 dilution of the i-ab=it or the pig serum. With these optimal dilutions the OD values for the positive pS4 plants ;arr~e:~
from 0.3 to 0.9. The OD of the negative plant antigen remained under O.l .
The plants that were found positive in ELISA were analyzed in western blo ttirlg so determine the size of the transgenic protein. The results for tobacco are shown u: FigLre 4. :'. fl transgenic plants expressed the protein of the expected size. The two protein bands in nS4 ~1>~.-:s represented glycosylated and ron ~giycosyl;~ted forms of the trans~cnic protein.
Direct measurement of the levels of transge=tic protein production was not carried cut ';~.-_ ~::::3 comparisons to known amounts of baculovinls expressed S genes and ELIS~a titrLtian:: i~;e ;~:_:_~~
that 0.1-0.2% of the total soluble leaf protein in tobacco was indeed the tra;~st~cruc prt;tela:. ~ua«r levels were found in alfal fa.
Antibody response oJswine to the transgenic proteins t)f;. I ) :~49 f~RJ 12: 47 F':1\ 1 i i S~ 821 ~W';f6 !WI i l'1: \ . !' RFSf:
ifR'Fl ;, Leaf extracts of the transgenic tobacco plants were used to immunize pya. ~?
Y. ~~ jr..~.:: ",-_., control groups were included, one immunized with wild type tobacco leaf e;aract ~:;.;s one,y::~~~:_.
with PBS instead of the plant extract. Blood samples were taken weekly and tl-~e I'~~.'r' s-~c:c= ;:
antibody levels were measured in virus neutralization assays and ELISA. fable 1. .l=o>=v t':.~ ~--::
and ELISA titers of the individual pigs detected in the serum samples ;
~:l::ctv~ ~:~~ ~~-s:i :: ~ - ..
experiment (week 6).
Both types of transgenic plant proteins were capable of induc.in~ st;ang 'I'i;E Y ,r:~Li=..-..
immune response as measured by the ELISA and the same sera also cliciteu VAT
acti. ity al~ho::_ to at a lower level.
Table 1. VN and ELISA titers of the individual blood samples collected at the bt"li we~.h. .';~~
indicates that there was no TGEV specific VN antibody in undiluted samples and no detecta~:' antibody in ELISA in 1:20 dilution). -t5 Plant Pig VN EIA

851 1:2 1:80 pSS 1 852 1:4 1:160 854 1:2 1:80 874 Killed the first after week pS4 875 1:8 1:320 B3 1:4 1:160 _ Wild type 856 0 0 No plant 858 0 G

While the present application has been described with reference to wh=' are p;~s;:.nt'=v considered to be the preferred examples, it is to be understood that the invention is f:ot lira=c~
the disclosed examples. To the contrary, the invention is intended to cover various mf~dii3;:ati~s:a 2o and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by' r:';erc;:=~ :, their entirety to the same extent as if each individual publication, patent or patent apr~lraat~;:t;,'_~
specifically and individually indicated to be incorporated by reference in its entirety.

~;5 I I - 99 H'R f L2: 48 F:1.1 I :~ I !? ~i21 :i'~36 r)I I I ('I~: V. I'.
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plant cells: an essential tool for antibody production and immunomodulation al° t:lezsi:~=,~;:~:
functions and pathogen activity. Plant Mol Biol 1998 Sep;38(1-2):101-9.
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to Enjuanes L Localization of antigenic sites of the E2 glycoprotein of iransmissibl~ Ccistre~.r_W~:s coronavirus. J Gen Virol 1990 Feb;71 ( Pt 2):271-9.
Delmas B, Rasschaert D, Godet M, Gelfi J, Laude IJ Four major anti;Ltzic sir~:.s of t;:e c.orr_~ra~:i'~;_, transmissible gastroenteritis virus are located on the amino-tetzninal half of sake gl~;ccnroe:, ~.
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gastroenteritis virus neutralization. J Virol 1986 Oct;60(1):131-9.
35 Koziel MG, Carozzi IrrB, Desai N Optimizing expression of transgenes with an emphasis c:r~ p_~:_ transcriptional events. Plant Mol Biol 1996 Oct;32(1-2):393-405.

~;ti- I 1 ~ 99 FRI 12: 48 F.a1 1 ~~ i ~~ 521 :W;sh fl ; l c'i: ' . I'.
KI~:Sf~;.IRC:l1 Laemmli UK Cleavage of structural proteins during the assembly of the he:a~'..
o~ 1;4:x.;.-::y :;-::~:~_~:. ;
Nature 1970 Aug 15;227(259):680-5.
Ni M. et al., Plant J 1995, 7:661.
Ni M, Cui D, Gelvin SB Sequence-specific interactions of wound-inducible rm~.l..a.s ~vt:,as ;~...
mannopine synthase 2' promoter wound-recpt~nsive elements. Plant htcvl T~~:;i 199 '_:r,_;;~; a :: -~ .
Sambrook F., Fritsch E.., lta~tiatis T. Vlolccmar Cloning: :~ Lai'aorato:y ..'v~~ru~;:~l, ,'' ~'~i;:-._-, Spring Harbour Laboraton.~ Press, Cold Spring Ii3rbo',ir, New York, 1 ~~ ~ ~.
Smerdou C, Anton IM, Plana J, Curtiss R 3rd, Enjuanes L ,A continuous epitapv uor:r t:~r:~:n--;-.--_.-.
gastroenteritis virus S protein fused to E. coli heat-labile toxin B subunit ~xprcssed b y attL'a__ to Salmonella induces serum and secretory immunity. Virus Res I99~ Mar;.~I(i j: (-9.
Smerdou C, Umiza A, Curtis R III, Enjuanes L Characterization of tr~~s~;i;ssihd~ gf:~rJ~~.;:;riT:
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persistence, stability and immune response in swine. Vet Microbiol 1996 Jan;48(1-2):87-l~~(I.
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Tuboly T, Nagy E, Dennis JR, Derbyshire JB Immunogenicity of the S protein of trars.~.i~i~~;=
2o gastroenteritis virus expressed in baculovirus. Arch Virol 1994; 137(1-2):SS-67.
Tuboly T, Nagy E, Derbyshire JB Potential viral vectors for the stimulation of mucosal arzt.i',acn responses against enteric viral antigens in pigs. Res Vet Sei 1993 May;S4(3):345-S0.
Van Kan et al. Plant Mol. Biol. 1989, 12:IS3-5.

. CA 02272793 1999-12-10 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: University of Guelph (ii) TITLE OF INVENTION: Porcine Transmissible Gastroenteritis Virus Oral Vaccine Production in Plants (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS
(A) NAME: Cowling, Strathy & Henderson (B) STREET: 160 Elgin Street, Suite 2600 (C) CITY: Ottawa (D) PROVINCE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE: K1P 1C3 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2,272,793 FILING DATE: June 11, 1999 CLASSIFICATION:
(vii) PATENT AGENT INFORMATION:
(A) NAME: COWLING, STRATHY & HENDERSON
(B) TELEPHONE: (613) 233-1781 (C) TELEFAX: (613) 563-9869 (D) REFERENCE NUMBER: 08-883769CA
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1856 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA; artificial sequence based on TGEV S-gene, modified for optimized codon usage for plant expression"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 610 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (A) DESCRIPTION: /desc = "Artificial sequence: Synthetic sequence based on TGEV S-gene, optimized codon usage for plant expression"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
Met Ala Phe Leu Lys Ser Phe Pro Phe Tyr Ala Phe Leu Cys Phe Gly Gln Tyr Phe Val Ala Val Thr His Ala Asp Asn Phe Pro Cys Ser Lys Leu Thr Asn Arg Thr Ile Gly Asn Gln Trp Asn Leu Ile Glu Thr Phe Leu Leu Asn Tyr Ser Ser Arg Leu Pro Pro Asn Ser Asp Asp Val Leu Gly Asp Tyr Phe Pro Thr Val Gln Pro Trp Phe Asn Cys Ile Arg Asn Asn Ser Asn Asp Leu Tyr Val Thr Leu Glu Asn Leu Lys Ala Leu Tyr Trp Asp Tyr Ala Thr Glu Asn Ile Thr Trp Asn His Arg Gln Arg Leu Asn Val Val Val Asn Gly Tyr Pro Tyr Ser Ile Thr Val Thr Thr Thr Arg Asn Phe Asn Ser Ala Glu Gly Ala Ile Ile Cys Ile Cys Lys Gly Ser Pro Pro Thr Thr Thr Thr Glu Ser Ser Leu Thr Cys Asn Trp Gly Ser Glu Cys Arg Leu Asn His Lys Phe Pro Ile Cys Pro Ser Asn Ser Glu Ala Asn Cys Gly Asn Met Leu Tyr Gly Leu Gln Trp Phe Ala Asp Glu Val Val Ala Tyr Leu His Gly Ala Ser Tyr Arg Ile Ser Phe Glu Asn Gln Trp Ser Gly Thr Val Thr Phe Gly Asp Met Arg Ala Thr Thr Leu Glu Val Ala Gly Thr Leu Val Asp Leu Trp Trp Phe Asn Pro Val Tyr Asp Val Ser Tyr Tyr Arg Val Asn Asn Lys Asn Gly Thr Thr Val Val Ser Asn Cys Thr Asp Gln Cys Ala Ser Tyr Val Ala Asn Val Phe Thr Thr Gln Pro Gly Gly Phe Ile Pro Ser Asp Phe Ser Phe Asn Asn Trp Phe Leu Leu Thr Asn Ser Ser Thr Leu Val Ser Gly Lys Leu Val Thr Lys Gln Pro Leu Leu Val Asn Cys Leu Trp Pro Val Pro Ser Phe Glu Glu Ala Ala Ser Thr Phe Cys Phe Glu Gly Ala Gly Phe Asp Gln Cys Asn Gly Ala Val Leu Asn Asn Thr Val Asp Val Ile Arg Phe Asn Leu Asn Phe Thr Thr Asn Val Gln Ser Gly Lys Gly Ala Thr Val Phe Ser Leu Asn Thr Thr Gly Gly Val Thr Leu Glu Ile Ser Cys Tyr Thr Val Ser Asp Ser Ser Phe Phe Ser Tyr Gly Glu Ile Pro Phe Gly Val Thr Asp Gly Pro Arg Tyr Cys Tyr Val His Tyr Asn Gly Thr Ala Leu Lys Tyr Leu Gly Thr Leu Pro Pro Ser Val Lys Glu Ile Ala Ile Ser Lys Trp Gly His Phe Tyr Ile Asn Gly Tyr Asn Phe Phe Ser Thr Phe Pro Ile Asp Cys Ile Ser Phe Asn Leu Thr Thr Gly Asp Ser Asp Val Phe Trp Thr Ile Ala Tyr Thr Ser Tyr Thr Glu Ala Leu Val Gln Val Glu Asn Thr Ala Ile Thr Lys Val Thr Tyr Cys Asn Ser His Val Asn Asn Ile Lys Cys Ser Gln Ile Thr Ala Asn Leu Asn Asn Gly Phe Tyr Pro Val Ser Ser Ser Glu Val Gly Leu Val Asn Lys Ser Val Val Leu Leu Pro Ser Phe Tyr Thr His Thr Ile Val Asn Ile Thr Ile Gly Leu Gly Met Lys Arg Ser Gly Tyr Gly Gln Pro Ile Ala Ser Thr Leu Ser Asn Ile Thr Leu Pro Met Gln Asp His Asn Thr Asp Val Tyr Cys Ile Arg Ser Asp Gln Phe Ser Val Tyr Val His Ser Thr Cys Lys Ser Ala Leu Trp Asp Asn Ile Phe Lys Arg Asn Cys Thr Asp His His His His His His

Claims (26)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OF PROVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of preparing a porcine transmissible gastroenteritis virus (TGEV) protein comprising the steps of:
(a) obtaining a S gene for TGEV that has been altered for optimal expression in the plant;
(b) inserting the gene within a vector suitable for transforming a plant;
(c) transforming the plant with the vector; and (d) growing the transformed plant.

2. The method according to claim 1, wherein the S gene for TGEV is truncated at the ~
end by approximately 600 base pairs.

3. The method according to claim 1, wherein the S gene has been resynthesized for optimal plant codon recognition,

4. The method according to claim 3, wherein the S gene is the nucleotide sequence in Figure 1.

5. The method according to claim 1, wherein the plant is selected from the group consisting of tobacco, alfalfa, barley, corn, flax, soybean, sunflower, rapeseed and wheat.

6. The method according to claim 5, wherein the plant is selected from harvested alfalfa.

7. The method according to claim 1, wherein the protein is extracted from harvested plant tissues and purified.

8. An isolated DNA molecule comprising the sequence defined it Figure 1.

9. A vector comprising the DNA molecule of claim 8, operatively associated with a promoter, enhancer and terminator regions.

10. A plant comprising the vector of claim 9.

11. A vector comprising a TGEV S gene that has been truncated at the 3' end by 600 bu~~
pair operatively associated with promoter, enhancer and terminator regions.

12. A plant comprising the vector of claim 11.

13. A protein produced by the method of claim 3.

14. A method of immunizing pigs against transmissible gastroenteritis virus comprising the steps of:
(a) transforming a plant with a vector selected from the group consisting of the vector of claim 9 and the vector of claim 11;
(b) growing the transformed plant;
(c) feeding tissues of the transformed plant to pigs; and (d) repeating step (c) as needed.

15. The method of claim 14, wherein the step of feeding the tissues of the transformed plant to pigs comprises grazing unharvested tissues.

16. The method of claim 14, wherein prior to step (c), tissues of the plant are harvested and the harvested tissues are fed to pigs.

17. The method of claim 14, wherein prior to step (c), tissues of the plant are harvested and the harvested tissue is extracted and the extract is used to immunize a pig.

18. A method of immunizing pigs against transmissible gastroenteritis virus comprising the steps of:
(a) transforming a plant with a vector selected from the group consisting of the vector of claim 9 and the vector of claim 11;
(b) growing the transformed plant;
(c) harvesting the transformed plant to obtain harvested tissue;
(d) extracting the protein encoded by the vector from the harvested tissue;

(e) administering the protein to the pig; and (f) repeating step (e) as needed.

19. The method of claim 18, wherein in step (c), the protein is administered to the pig ~~~
food supplement or as an injection.

20. The method of claim 1, wherein the tissues are harvested from the transformed plant to obtain harvested tissue.

21. The method of claim 20, wherein the protein is extracted from the harvested tissue.

22. An animal feed composition comprising a tissues from a transgenic plant, wherein the transgenic plant comprises a vector selected from the group consisting of the vector of claim 9 and the vector of claim 11.

23. An immunogenic composition comprising tissues from a transgenic plant having a vaccine antigen that provides for protection against porcine TGEV and which is encoded by a vector selected from the group consisting of the vector of claim 9 and the vector of claim 11.

24. The composition according to claim 23, further comprising an adjuvant.

25. A method for protecting a pig againt porcine TGEV comprising administering orally an immunogenic composition according to claim 23 in an amount effective to provide protection against porcine TGEV to a pig.

26. The method according to claim 25, wherein the immunogenic composition is administered by feeding it to a pig.